The proposed research program will focus on the human platelet alpha-adrenergic receptor. This receptor will be characterized directly using ligand binding assays. A major objective is to develop an agonist ligand binding assay for the receptor using (3H) epinephrine. Preliminary evidence has been obtained demonstrating the feasibility of this objective. Using pyrocatechol (1 mM), ascorbic acid 0.004% and pargyline (1 micromole) to protect the ligand, we have demonstrated specific binding which is saturable and stereospecific for catecholamines. Catecholamines compete for binding with the alpha-adrenergic potency series. (3H)-Epinephrine binding data will be compared and contrasted with data using the ligand (3H)-dihydroergocryptine, an alpha-adrenergic antagonist. The Kd of (-) epinephrine for the platelet alpha-adrenergic receptor determined form (3H)-epinephrine binding is 30nM or 1 1/2 log units lower than that obtained (3H)-dihydroergocryptine. These data suggest that physiologic concentrations of catecholamines may interact with the alpha-adrenergic receptor and modify platelet function. Data using both ligands will be correlated with catecholamine induced aggregation, inhibition of adenylate cyclase, and potentiation of aggregation. New semi quantitative techniques for measuring aggregation potentiation are proposed. Ligand and biochemical regulation of the alpha-adrenergic receptor will be studied. Agonist induced desensitization of the platelet alpha-adrenergic receptor has been demonstrated and will be characterized more extensively both in intact platelets and in platelet membranes. In addition, the alpha-adrenergic receptor will be solubilized and the soluble receptor characterized using (3H)-epinephrine binding. Preliminary data indicate that Triton X-100 can adequately solubilize the receptor. These studies will be extended to the evaluation of alpha-adrenergic receptor function in patients exhibiting abnormalities in catecholamine-induced aggregation. These studies will provide new insight into the basic biochemistry and physiology of alpha-adrenergic platelet and into the potential role of catecholamines in modulating platelet functions. The proposal will also contribute to the understanding of the basic physiology and biochemistry of the alpha-adrenergic receptor in a more general fashion. The proposed research will con (Text Truncated - Exceeds Capacity)